Identification of ATP-NADH kinase isozymes and their contribution to supply of NADP(H) in Saccharomyces cerevisiae
Identification of ATP-NADH kinase isozymes and their contribution to supply of NADP(H) in Saccharomyces cerevisiae
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DOI：
10.1111/j.1742-4658.2005.04749.x
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发表时间：
2005-07-01
期刊：
影响因子：
5.4
通讯作者：
Murata, K
中科院分区：
文献类型：
--
作者：
Shi, F;Kawai, S;Murata, K
ATP-NAD激酶通过使用ATP磷酸化NAD以产生NADP，而ATP-NADH激酶磷酸化NAD和NADH。在酿酒酵母（Saccharomyces cerevisiae）中发现了三种NAD激酶同源物，即ATP-NAD激酶（Utr 1 p）、ATP-NADH激酶（Pos 5 p）和功能未知的Yel 041 wp（Yef 1 p）。在本研究中，Yef 1 p被鉴定为ATP-NADH激酶。Utr 1 p的ATP-NADH激酶活性也得到了证实。因此，这三种NAD激酶同源物经生化鉴定为ATP-NADH激酶。对UTR 1、YEF 1和POS 5的单突变体、双突变体和三突变体的表型分析（出乎意料地发现是可行的）证明了Pos 5 p对线粒体功能和37 ℃下的存活的关键贡献，以及Utr 1 p对低铁培养基中生长的关键贡献。其他两种酶的贡献也得到了证实;然而，这些仅在关键贡献者不存在的情况下观察到，这得到了UTR 1和YEF 1过表达对某些pos 5表型的互补的支持。三重突变体的生存能力表明，一种“新型”酶可能催化S中细胞质NADP的形成，该酶的一级结构与所有已知的NAD和NADH激酶的一级结构不同。啤酒。最后，我们发现光滑假丝酵母LEU 2编码β-异丙基苹果酸脱氢酶，并用于构建三重突变体，补充了一些pos 5表型; cerevisiae没有。这种互补作用主要是由于C. glabrata使用NADP作为辅酶并提供NADPH。
ATP-NAD kinase phosphorylates NAD to produce NADP by using ATP, whereas ATP-NADH kinase phosphorylates both NAD and NADH. Three NAD kinase homologues, namely, ATP-NAD kinase (Utr1p), ATP-NADH kinase (Pos5p) and function-unknown Yel041wp (Yef1p), are found in the yeast Saccharomyces cerevisiae. In this study, Yef1p was identified as an ATP-NADH kinase. The ATP-NADH kinase activity of Utr1p was also confirmed. Thus, the three NAD kinase homologues were biochemically identified as ATP-NADH kinases. The phenotypic analysis of the single, double and triple mutants, which was unexpectedly found to be viable, for UTR1, YEF1 and POS5 demonstrated the critical contribution of Pos5p to mitochondrial function and survival at 37 degrees C and the critical contribution of Utr1p to growth in low iron medium. The contributions of the other two enzymes were also demonstrated; however, these were observed only in the absence of the critical contributor, which was supported by complementation for some pos5 phenotypes by the overexpression of UTR1 and YEF1. The viability of the triple mutant suggested that a 'novel' enzyme, whose primary structure is different from those of all known NAD and NADH kinases, probably catalyses the formation of cytosolic NADP in S. cerevisiae. Finally, we found that LEU2 of Candida glabrata, encoding beta-isopropylmalate dehydrogenase and being used to construct the triple mutant, complemented some pos5 phenotypes; however, overexpression of LEU2 of S. cerevisiae did not. The complementation was putatively attributed to an ability of Leu2p of C. glabrata to use NADP as a coenzyme and to supply NADPH.